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United States Patent |
6,151,519
|
Sugihara
,   et al.
|
November 21, 2000
|
Planar electrode
Abstract
A planar electrode which enables multi-point simultaneous stimulation and
recording of nerve cells over a long time is provided, which also has
excellent response property. An ITO film is evaporated on the whole
surface of an insulating substrate of the hard glass, and the ITO film is
etched so that the central portion of each electrode is located on each
intersection of 8.times.8 lattices, the center-to-center distances of
nearest electrodes of each electrode are equal, and a lead wire is
stretched radially. Then, for an insulating layer, negative
photo-sensitive polyimide is spin-coated, and an insulating layer pattern
is exposure-formed so that a 50 .mu.ms square hole is produced at the
center of each electrode. Furthermore, to the exposed portion of each
electrode (that is, the inside of the 50 .mu.ms square), Ni is evaporated
in a film thickness of 500 nm, which is followed by evaporating gold (50
nm) and platinum black (about 1 .mu.m). The contact with the external
circuit of the section near the end opposite to the electrode of the lead
wire was coated with gold and nickel.
Inventors:
|
Sugihara; Hirokazu (Katano, JP);
Taketani; Makoto (Kyoto, JP);
Mitsumata; Tadayasu (Hirakata, JP)
|
Assignee:
|
Matsushita Electric Industrial Co., Ltd. (Osaka, JP)
|
Appl. No.:
|
160252 |
Filed:
|
September 22, 1998 |
Foreign Application Priority Data
Current U.S. Class: |
600/372; 204/403.01; 435/287.1; 600/373; 607/116 |
Intern'l Class: |
A61B 005/04 |
Field of Search: |
600/372,373
607/116,117,148
204/403
435/287.1
|
References Cited
U.S. Patent Documents
5810725 | Sep., 1998 | Sugihara et al. | 600/372.
|
Primary Examiner: Cohen; Lee
Attorney, Agent or Firm: Morrison & Foerster LLP
Parent Case Text
This application is a continuation of U.S. Ser. No. 08/481,149, filed Jun.
7, 1995, now U.S. Pat. No. 5,810,725, which is a continuation-in-part of
U.S. Ser. No. 08/114,634, filed Sep. 2, 1993, now abandoned.
Claims
What is claimed is:
1. A planar electrode having a multiplicity of microelectrodes for
measurement of electric activities of nerve cells, comprising an electrode
section having a multiplicity of microelectrodes insulated from each other
and each connectable to outside said electrode section, said
microelectrodes each having an area from 4.times.10.sup.2 .mu.m.sup.2 to
4.times.10.sup.4 .mu.m.sup.2, wherein said microelectrodes have an
impedance in the range of 1 .OMEGA. to 100 k.OMEGA. when said electrode
section is covered with an electrolyte solution and an alternating voltage
of 1 kHz, 50 mV is provided to a reference electrode between said
electrolyte solution and said microelectrodes.
2. The planar electrode of claim 1 wherein each of said microelectrodes has
a coating thereon.
3. The planar electrode of claim 2 wherein each of said microelectrodes is
comprised of an indium tin oxide alloy, covered thereon with at least one
metal selected from the group consisting of Ag, Al, Bi, Au, Cu, Cr, Co,
and Ni, and coated thereon with a metal containing platinum.
4. The planar electrode of claim 2 wherein each of said microelectrodes is
comprised of an indium tin oxide alloy, covered thereon with Au and Ni,
and coated thereon with a metal containing platinum.
5. The planar electrode of claim 2 wherein the multiplicity of
microelectrodes are spaced apart with a microelectrode-to-microelectrode
distance in the range of 10 .mu.m to 1000 .mu.m.
6. The planar electrode of claim 2 wherein a solution holding section for
holding a cell culture liquid medium surrounds said electrode section.
7. The planar electrode of claim 2 wherein the multiplicity of
microelectrodes are arranged in an 8.times.8 lattice.
8. The planar electrode of claim 2 wherein said impedance is in the range
of 1 k.OMEGA. to 60 k.OMEGA. when an alternating voltage of 1 kHz, 50 mV
is provided to a reference electrode between said electrolyte solution and
said microelectrodes.
Description
FIELD OF THE INVENTION
This invention relates to a planar electrode which is used in the field of
neurophysiology for electric measurement of biological activities, in
particular, of the electric activities of nerve cells. This planar
electrode comprises a large number of electrodes.
BACKGROUND OF THE INVENTION
Recently, medical investigations on nerve cells and investigations of the
possibility of using nerve cells as electric elements have been actively
pursued. When nerve cells are active, action potential is generated. The
action potential is generated by the change of ion concentration inside
and outside the cell membrane which is accompanied by the change of ion
permeability in nerve cells. Measuring this potential change accompanied
by the ion concentration change (that is, the ion current) near the nerve
cells with electrodes enables the detection and investigation of nerve
activities.
Conventionally, in order to measure the electrical activities of nerve
cells, it is common practice to use a recording electrode comprising glass
or other electrodes and a stimulating electrode comprising metal or other
electrodes, insert each of them in or between cells, and measure the
electrical activities of nerve cells with the recording electrode when a
stimulating current (or voltage) is applied from the stimulating
electrode.
In addition to this, there are many modified methods such as the so-called
patch clamp method, in which a cell body is pierced with a capillary glass
suction electrode, the inside of the cell body is refluxed with the liquid
in the glass suction electrode, and electrical signals are emitted from
this glass suction electrode to observe electric characteristics of the
cell membrane.
Furthermore, a method of recording electrical activities of nerve cells is
proposed separately from the inventors of this invention, which is
accomplished by forming electrodes made of a conductive material such as
ITO (indium tin oxide) on the surface of an insulating substrate with a
diameter of 15 to 20 .mu.m, enabling culture of nerve cells on the
electrodes, and applying electric stimulation to cells without piercing
the cells with electrodes.
As an improvement of this method, the inventors of this invention also
propose separately to form electrodes with a diameter of 20 to 200 .mu.m,
so that an electric potential difference arising between the electrodes
becomes smaller when constant current stimulation is applied to nerve
cells. As a result, ITO is less likely to be destroyed, thereby enabling
even more long-term observation.
In the above-mentioned conventional technique and its modified methods,
electrodes such as glass electrodes, which have to be larger than the
cells themselves, must be urged. As a result, primarily due to
restrictions of space and operating accuracy, multi-point simultaneous
measurements in which two or more recording electrodes are inserted
simultaneously in one sample to record electrical activities of the nerve
cells are extremely difficult.
In order to investigate the operation of the whole nerve circuit network,
it is necessary to record many nerve cell activities simultaneously, and
as the number of measuring points increases, the degree of difficulty
increases, creating the problem that it is difficult to observe throughout
a large number of cells.
In addition, because glass, metal, or other electrodes must be pierced into
or between cells, there is another problem that the damage to the cell is
serious and measurement over a long time such as extending for more than a
few hours is difficult to carry out.
On the other hand, signal transmission throughout a large number of cells
can be observed by using an insulating substrate formed thereon with
circular (or square) electrodes made of a conductive material such as ITO
with a diameter (or a side) of 15 to 20 .mu.m. However, due to the small
area of the electrodes ranging from 177 .mu.m.sup.2 to 400 .mu.m.sup.2,
the electrode resistance at the interface of culture solution becomes
several M.OMEGA.. Since the stimulation is generally provided as constant
electric current, an extremely large potential difference arises between
the electrodes when the electric resistance is large. Thus, ITO is
destroyed when a long-term electric stimulation is provided under such
large voltage, creating the problem that it is difficult to carry out
observations over a long time.
In addition, when the electrode area ranges from 400 .mu.m.sup.2 to 40000
.mu.m.sup.2, the electrode resistance at the interface of culture solution
is reduced, and a potential difference arising between the electrodes
becomes comparatively small. Even if the stimulating electric current was
provided over a long time, destruction of ITO was not observed by a
microscope. However, when a stimulating current was applied at a certain
electrode and a potential change accompanied by the stimulation was
recorded at other electrodes, a great change was observed in the recording
waveform before and after long stimulation. In other words, the effects of
the applied stimulating current on the recording waveform (that is,
artifacts) were greater after long stimulation than before long
stimulation. The reason for this waveform change is considered to be
caused by polarization on the electrode surface. In the worst case, the
electrical activities of the nerve cells were hidden by the artifacts and
measurement was disabled. Furthermore, even if the artifacts are not so
great, there was another problem that it becomes difficult to compare
strength of nerve activities before and after long stimulation.
SUMMARY OF THE INVENTION
Accordingly, a feature of the invention is to provide a planar electrode
which solves these conventional problems and enables easy multi-point
simultaneous stimulation and measurement of nerve cells as well as signal
transmission and observation throughout many cells for more than just a
few hours. The planar electrode of the invention also enables suppressing
generation of artifacts which is accompanied by applied stimulating
electric current, and comparison of potential recording waveforms before
and after long stimulation.
In order to accomplish these and other objects and advantages, a planar
electrode of this invention has a multiplicity of electrodes for
measurement of electric activities of nerve cells in an organism, and
comprises an insulating substrate, a multiplicity of electrodes disposed
thereon, a wiring section in which lead wires are installed from the
electrodes, and an insulating layer covering the lead wires, and the
electrodes each has an area from 4.times.10.sup.2 .mu.m.sup.2 to
4.times.10.sup.4 .mu.m.sup.2, wherein impedance is in the range of 1
.OMEGA. to 100 k.OMEGA. when the electrode section is covered with an
electrolytic solution and an alternating voltage of 1 kHz, 50 mV is
provided to an optional portion between the electrolytic solution and the
lead wires.
It is preferable in this invention that the lead wire comprises an indium
tin oxide alloy. Accordingly, continuity can be established with
reliability even if the wire is thin. In addition, when an indium tin
oxide alloy is used for the lead wire, the lead wire becomes transparent
with a slightly yellowish color. This feature is preferable for attaining
good visibility of nerve cells and also for operations in experiments.
Furthermore, it is preferable in this invention that the electrode is
comprised of an indium tin oxide alloy, covered thereon with at least one
metal selected from the group consisting of Ag, Al, Bi, Au, Cu, Cr, Co,
and Ni, and coated thereon with a metal containing platinum. This
structure enables establishment of reliable continuity with low impedance.
In addition, even if the electrode comes into contact with an electrolyte,
no alternation such as oxidation occurs.
In addition, it is preferable in this invention that the lead wire drawer
section is comprised of an indium tin oxide alloy, covered thereon with at
least one metal selected from the group consisting of Ag, Al, Bi, Au, Cu,
Cr, Co, and Ni. This structure can attain strong oxidation resistance and
is convenient to take out electric signals from electrodes.
Also, when the shortest electrode-to-electrode distance between adjacent
electrodes in the multiplicity of electrodes is substantially equal in
this invention, it is convenient to measure electric stimulation signals
of nerve cells etc. Here, the electrode-to-electrode distance indicates
the distance of the closest parts in the electrodes.
When the electrode-to-electrode distance is in the range of 10 .mu.m to
1000 .mu.m in this invention, it is similarly convenient to measure
electric stimulation signals of nerve cells etc.
It is preferable in this invention that the lead wires are installed
substantially radially from the electrodes to reduce the capacitance
between the lead wires.
Furthermore, it is preferable in this invention that a solution holding
section for holding a cell culture liquid medium is present in a position
surrounding a group of multielectrodes.
Thus, measurements can be performed while culturing cells for a long period
of time.
In addition, it is preferable in this invention that the insulating layer
covering the lead wires has holes above each electrode and is provided
approximately on the entire surface of the insulating substrate except in
the vicinity of contact points between an external circuit and the lead
wires. In this way, even if an electrolytic solution or a culture liquid
medium is dropped, the lead wires are not directly touched and remain
electrically stable. In particular, the insulating layer comprising a
polyimide resin or an acrylic resin is chemically and thermally stable.
Also, when centers of the multiplicity of electrodes are located at each
intersection of an 8.times.8 lattice in this invention, it is practically
sufficient to measure nerve cells in an organism.
The electrolytic solution comprising a NaCl aqueous solution of 1.4 vol. %
is close to a cell culture liquid medium. Since electric conductivity is
close to a cell culture liquid medium actually used for measurments of
nerve cells in an organism, it is suitable for measuring impedance when an
alternating current is passed to an optional section between the
electrolytic solution and the lead wires. This is because measurements can
be conducted under the conditions which are close to an environment to be
used.
In addition, it is practical that impedance is in the range of 1 k.OMEGA.
to 60 k.OMEGA. when an alternating voltage of 1 kHz, 50 mV is provided to
an optional portion between the electrolytic; solution and the lead wires.
The planar electrode of the invention enables detection of the transmission
of signals between adjoining cell bodies in providing signals to nerve
cells cultured on the planar electrode of the invention and measuring the
signal between cells at the same time. This is because one cell body is
arranged on the electrode, and it can be arranged with a high degree of
probability that the cell body mediating the cell protrusions (e.g.,
dendrites and axons on nerve cells) will be located on adjoining
electrodes by adjusting the shortest electrode-to-electrode distance to be
nearly equal to the length of a nerve cell to be measured (that is, cell
body, dendrites, axon) and equally spacing the electrodes.
Furthermore, arranging the lead wires extending from the electrodes
substantially radially reduces the capacitive component (capacitance)
between lead wires from the capacitance when they are arranged in
parallel. The collapse of pulse signal waveform, electrical signals, is
also reduced, and the time constant of the circuit becomes small,
improving the response to quick pulse signals and therefore improving the
follow-up to the component with fast nerve cell activities.
In addition, adjusting the electrode area in the range from
4.times.10.sup.2 to 4.times.10.sup.4 .mu.m.sup.2 enables application of
electric stimulation to a cell over a long time extending for more than a
few hours as well as measurement of electric activities of the cell.
Determining the impedance to achieve the desired range of 1 .OMEGA. to 100
k.OMEGA. substantially prevents polarization on the stimulating electrode
surface of the nerve cell from occurring when a stimulating current is
applied for a long time to nerve cells at a certain electrode and
electrical activities (potential change) of the nerve cells are recorded
corresponding to the stimulating current at other electrodes, thereby
minimizing the effects (that is, artifacts) of stimulating current on
potential recording waveform. In particular, because even after a
stimulating current is applied over a long time, artifacts are small and
the mode is free from change, so electrical activities of nerve cells
before and after long stimulation can be compared.
In addition, in the planar electrode of the invention, bringing the
shortest electrode-to-electrode distance to the desirable condition of 10
to 1000 .mu.m results in a high possibility of locating the cell bodies on
adjoining electrodes and connecting them via their axons, since nerve
cells generally have axons with a length within this range under culture
conditions, achieving an electrode-to-electrode distance convenient for
measurement of nerve cells.
In the planar electrode of the invention, the desirable form of the
insulating layer, in which the insulating layers covering lead wires have
holes over each electrode and are installed on nearly the whole surface of
the insulating substrate except the vicinity of the section where the lead
wire comes in contact with the external circuit, allows easy formation of
the required insulating layer by applying the insulating material
comprising photo-sensitive resin to nearly the whole surface and removing
the insulating layer on each electrode by a photoetching method and
opening holes to expose electrodes, thereby achieving easy production and
minimizing the probability of insulation failure, which is very desirable.
This process is superior to optionally forming insulating layers merely on
lead wires.
Furthermore, in the planar electrode of the invention, locating the center
of a multiplicity of electrodes at each intersection of 8.times.8 lattices
secures the maximum number of electrodes which enables installation of
lead wires substantially radially from the electrodes of the invention,
which is very desirable.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plan view showing a pattern in the center of a wire section
prior to the application of an insulating layer on the planar electrode of
the invention with electrodes and lead wires formed on an insulating
substrate in an embodiment of this invention;
FIG. 2 is a partially notched view of the plan view of FIG. 1 having the
insulating layer of an embodiment of the invention applied to a planar
electrode;
FIG. 3 is a fragmentary cross sectional view of an embodiment of a planar
electrode of the invention;
FIG. 4 shows a changing potential waveform recorded with other suitable
electrodes before applying stimulating current over a long time using
suitable electrodes in an embodiment of the planar electrode of the
invention;
FIG. 5 shows the changing potential waveform recorded with other suitable
electrodes after applying stimulating current over a long time using
suitable electrodes in an embodiment of the planar electrode of the
invention;
FIG. 6 shows the changing potential waveform recorded with other suitable
electrodes before applying stimulating current over a long time with
suitable electrodes, using a planar electrode which differs from the
planar electrode of the invention only in that the electrode surface is
not coated with Ni, Au, and platinum black;
FIG. 7 shows another kind of the changing potential waveform recorded with
electrode after applying stimulating current over a long time with
suitable electrodes, using a planar electrode which differs from the
planar electrode of the invention only in that the electrode surface is
not coated with Ni, Au, and platinum black;
FIG. 8 is a fragmentary cross sectional view showing a method of measuring
impedance in an embodiment of the planar electrode of this invention.
DETAILED DESCRIPTION OF THE INVENTION
As the insulating substrate material used for the invention, a transparent
substrate is desirable because microscopic observation is required
throughout the experiment. Examples include glasses such as quartz glass,
lead glass or borosilicate glass, or inorganic substances such as quartz,
or organic substance with transparency such as polymethyl metacrylate or
its copolymers, polystyrene, polyvinyl chloride, polyester, polypropylene,
urea resin, and melamine resin. Considering mechanical strength in
combination with transparency, though, inorganic substances are desirable.
As the electrode materials used for the invention, examples include indium
tin oxide (ITO), tin oxide, Cr, Au, Cu, Ni and Al. ITO is particularly
desirable for its good conductivity.
The same materials can be applied to lead wires, and ITO is again desirable
for the similar reason mentioned above. In particular, by using the same
material for the lead wire section and the electrode section and
manufacturing them in a lump, the manufacturing process can be simplified
and the connection between the lead wire section and the electrode section
is even more assured.
It is not a particular restriction of the invention, but in general the
thickness of the electrodes and lead wires should be about 50-500 nm and,
in general, these materials are evaporated on the insulating substrate and
formed in a desired pattern by etching using a photoresist.
As the insulating layer material used to insulate the lead wires used for
the invention, examples include polyimide (PI) resin, epoxy resin, acrylic
resin, polyester resin, polyamide resin, and other transparent resins.
These types of resin are applied on lead wires by conventional techniques
to form an insulating layer. When the insulating material is a
photosensitive resin which has photochemical polymerization properties,
etc., it is desirable because patterns can be formed to provide holes on
the insulating layer portion on the electrodes to expose the electrodes as
described above.
In particular, when insulating material is PI or acrylic resin and the cell
to be cultured is a nerve cell, satisfactory growth takes place and
therefore, it is very desirable. In addition, among types of PI, negative
photosensitive polyimide (NPI) is most desirable because holes can be
formed on the electrodes using a photo etching process after the negative
photosensitive polyimide is applied over nearly the whole surface, in a
manner similar to that in forming patterns of the wiring section.
The thickness of the insulating layer may be such that can impart
insulating capability. This is not particularly limiting but in general, a
thickness of 0.1-10 .mu.m, specifically, one of 1-5 .mu.m is desirable.
The planar electrode of the invention was used to directly culture cells
and to measure and record electrical activities of the cells. Depending on
culture conditions or the type of cells, the size of cell body or the
length of cell protrusion such as dendrites or axons may vary but 10-1000
.mu.m is desirable for the electrode-to-electrode distance of the closest
planar electrodes. When the electrode-to-electrode distance is less than
10 .mu.m, the electrodes are so close to one another that the probability
for the cell bodies to adjoin via cell protrusions decreases. Furthermore,
wiring of lead wires become difficult. When the electrode-to-electrode
distance exceeds 1000 .mu.m, lead wires can be easily wired but as it is
rare for the cell protrusions to elongate as far as about 1000 .mu.m under
culture conditions, the probability of the cell bodys bonded via
protrusions and synapses located at the ends of the protrusions to be
located on the electrode decreases. Even under general culture conditions,
about 200-300 .mu.m is desirable for the electrode-to-electrode distance
because the length of cell protrusions of a cultured cell is about 200-300
.mu.m on average for central nervous system cells of mammals.
With respect to the electrode area, in order to avoid electrode breakage
which occurs when electric stimulation is applied to the nerve cells over
a long period of time and also to avoid excessive polarization, it is
necessary to reduce the impedance at the interface with the culture
medium, requiring a size exceeding a certain level.
Furthermore, when the impedance at the interface of the electrode and the
culture medium becomes smaller, thermal noise arising at this part is
reduced. On the other hand, a potential change accompanied by the
activities of the nerve cells which is detected by the electrode is
usually measured via an amplification circuit disposed outside. It is
common to use an amplification circuit having an extremely high input
impedance (for example, several hundreds M.OMEGA.) the value of the
measured potential change is hardly different, even if a slight change of
impedance (for example, change from several M.OMEGA. to several k.OMEGA.)
may be observed at the interface of the electrode and the culture medium.
Therefore, the signal to noise (S/IN) ratio of the measurement improves as
a whole, so that it is also advantageous from this aspect.
However, since the impedance at the interface of the electrode and the
culture medium is reduced, if the electrode area is enlarged too much, a
multiplicity of nerve cells must be placed on one electorde. In this case,
the potential change to be measured indicates the average activities of
the multiplicity of the nerve cells, so that it is extremely difficult to
analyze the activities of each nerve cell. Consequently, the electrode
area must be determined carefully according to the object of the
experiment.
In general, by taking the length of protrusion elongation of the nerve
cells under the above-mentioned culture conditions and other factors into
consideration, it is desirable that the electrode area ranges from
4.times.100 .mu.m.sup.2 to 4.times.10000 .mu.m.sup.2, and more preferably,
from 1.times.1000 .mu.m.sup.2 to 1.times.10000 .mu.m.sup.2.
In order to reduce the impedance between the electrode and an optional
point on the lead wire to be 100 k.OMEGA. or less when 1.4 vol. % NaCl is
filled on the electrode and an alternating voltage of 1 kHz, 50 mV is
provided, the ITO top surface is coated with metal. At least one of the
coating materials selected from the group consisting of Ag, Al, Bi, Au,
Cu, Cr, Co, and Ni can be used here, but with low toxicity to nerve cells
taken into account, the use of Au is desirable. The coating thickness is
not, in particular, limited, but is from 50 to 500 nm and, in general,
these materials are evaporated on the insulating substrate and are formed
into desired patterns by etching using a photoresist. When platinum black
is coated, corrosion-resistant property is improved, and at the same time,
the impedance is lowered, thereby also improving the conductivity.
In addition, according to a preferred embodiment of the invention described
previously, holes in the insulating layer of the planar electrode are
formed to expose electrodes not only to give electrical stimulation to the
cell body cultured on the planar electrode but also to detect electrical
activities from adjoining cell bodies which are located at the central
portion of the electrode.
Arranging lead wires stretched from the electrode nearly radially
eliminates the capacitance between lead wires, reduces the time constant,
and improves the measuring accuracy.
The configuration in which the electrode center portion of the planar
electrode of the invention is located concentrically or at each
intersection of lattices of 8.times.8 or smaller enables the lead wire to
be radially installed, and from the viewpoint of particularly forming as
many electrodes as possible and providing and recording multi-point
stimulation simultaneously, it is desirable to install electrodes at each
intersection of the 8.times.8 lattices.
Now referring the following specific embodiments, the planar electrode of
the invention will be described in further detail.
(Embodiment 1)
FIG. 1 is a plan view showing a pattern in the center of a wire section
prior to the application of an insulating layer on the planar electrode of
the invention with electrodes 1 and lead wires 2 formed on an insulating
substrate 3. FIG. 2 is a partially notched view of the plan view having
the insulating layer applied to a member shown in FIG. 1. FIG. 3 is a
fragmentary cross sectional view showing a planar electrode of this
invention. In FIG. 1, reference numeral 1 represents an electrode for
detection of electric activities of a cell body; 2 represents a lead wire
to be connected to the electrode 1; and 3 represents an insulating
substrate such as glass. In FIG. 2, 4 represents an insulating layer
comprising a polyimide resin etc., and 5 represents a hole opened in the
insulating layer 4 for forming electrodes. Next in FIG. 3, 1 represents a
planar electrode; 2 represents an ITO (indium tin oxide alloy) layer which
was formed on top of an insulating substrate 3 comprising glass etc.; 4
represents an insulating layer such as a polyimide resin; 6 represents a
layer formed on the surface of the ITO layer 2, for example, a Ni layer;
and 7 represents an Au layer formed thereon. 5 represents a foundation
electrode section comprising the Ni layer 6 and the Au layer 7 disposed
thereon. 8 represents a surface layer electrode section comprising, for
example, platinum black formed on the surface of the foundation electrode
section 5.
It will be explained in the following by referring to these figures.
First, fabrication of a planar electrode wiring section is described. As
the insulating substrate 3 of the planar electrode in FIGS. 1 and 3,
50.times.50.times.1 mm hard glass ("IWAKI CODE 7740 GLASS"--Iwaki Glass
Co., Ltd. or "CORNING 7059") was used; this is a transparent insulating
material with high mechanical strength. For the material of electrode 1
and lead wire 2, ITO was used, and on the whole surface of the insulating
substrate 3 of the hard glass, ITO was evaporated to form a layer about
1500 angstrom (150 nm) thick, which was followed by rinsing.
Then, the substrate was exposed to light through a photoresist so that the
central portion of each electrode 1 was located on each intersection of
8.times.8 lattices (position 5 as shown in FIG. 2), the center-to-center
distances of nearest electrodes of each electrode were equal, and lead
wire 2 formed the pattern of electrode I and lead wire 2 in which lead
wire 2 was stretched radially. It was then etched with ITO in a solution
which was made up using demineralized water, hydrochloric acid, and nitric
acid in a volume ratio of 50:50:1, and the photoresist was removed. The
wiring portion with electrode 1 being 60 .mu.ms square, lead wire 2 being
30 .mu.m wide, and a center-to-center distance of electrodes of 300 .mu.m
(that is, the electrode-to-electrode distance is 240 .mu.m) was thus
formed.
Then, for insulating layer 4, negative photo-sensitive polyimide
(hereinafter called "NIP") was spin-coated so that a film 1.4 .mu.m thick
was formed after drying, and an insulating layer pattern was
exposure-formed so that a 50 .mu.ms square hole 5 was produced at the
center of each electrode of the wiring section as shown in FIG. 2. As
shown in FIG. 3, to the exposed portion of each electrode (that is, the
inside of the 50 .mu.ms square), Ni 6 was evaporated to form a film of 500
nm thick, and on top of this Ni layer 6, gold 7 was evaporated to form a
film of 50 nm thick, thereby forming the foundation electrode section 5.
Contact points between the lead wires 2 and an external circuit of the
section near the end opposite to the electrode were also coated by
evaporating Ni 6 in a film thickness of 500 nm and gold 7 on top of the Ni
layer 6 in a film thickness of 50 nm to improve durability. The surface
layer electrode section 8 comprising a platinum black layer was formed on
the surface of the foundation electrode section 5 in the following method.
For the convenience of performing the coating process, a polystyrene
cylinder having an inner diameter 22 mm, an outer diameter 26 mm, and a
height 8 mm was adhered in the following steps. This polystyrene cylinder
can be sterilized and used as a culture container for culturing nerve
cells on a planar electrode.
(a) On the bottom face of a polystyrene cylinder (inner diameter 22 mm,
outer diameter 26 mm, height 8 mm) shown as 9 in FIG. 8, a sufficient
amount of an one-liquid silicon adhesive (DOW CORNING CO., LTD. 891 or
SHIN-ETSU CHEMICAL CO., LTD. KE-42RTV) was applied.
(b) The center of a glass substrate in the planar electrode and the center
of the polystyrene cylinder 9 were carefully matched and then adhered in
this condition.
(c) By leaving in an environment in which dusts hardly enter for 24 hours,
the adhesive was solidified.
The microelectrode surface was coated with platinum black in the following
steps.
(a) The above-mentioned polystyrene cylinder 9 was filled with 1 ml of
solution comprising 1 vol. % chloroplatinic acid, 0.0025 vol. % HCl, and
0.01 vol. % lead acetate (hereinafter abbreviated as a platinum solution).
(b) A platinum wire having a diameter of 0.2 mm was dipped in the platinum
solution up to a length of about 1 cm.
(c) By using the above-mentioned platinum wire as the positive electrode
and one of microelectrodes selected optionally as the negative electrode,
DC voltage was applied for 1 minute to pass electric current with current
density of 20 mA/cm.sup.2 between the two electrodes. According to this
operation, platinum black (3 in FIG. 8) of about 1 .mu.m thick was
deposited on the surface of the microelectrode serving as the negative
electrode.
(d) The operation of Step (c) was conducted for each microelectrode, so
that this operation was repeated for a total of 64 times.
(e) Alternatively, by shorting contact points from the microelectrodes via
the lead wires with an external circuit and by using them altogether as
the negative electrode, platinum black can be deposited on the surface of
a multiplicity of microelectrodes simultaneouly. At this time, however,
the DC voltage to be applied is adjusted to maintain the current density
of 20 mA/cm.sup.2.
The impedance was measured in the following steps.
(a) The above-mentioned polystyrene cylinder 9 was filled with 1 ml of 1.4
vol. % NaCl solution 10.
(b) A platinum wire 11 having a diameter of 0.2 mm was dipped in the 1.4
vol. % NaCl solution 10 up to a length of about 1 cm.
(c) By using the above-mentioned platinum wire 11 as one end and by
selecting the other end optionally from contact points 6, 7 from the
microelectrodes via the lead wires 2 with an external circuit, AC voltage
of 1 KHz, 50 mV was applied between the two ends, and impedance was
measured. The measurement was conducted by using a LCR meter (YHP 4274A).
As a result of the measurements, an average value of impedance was 10
k.OMEGA. when platinum black was present. On the other hand, the average
value of impedance was 500 k.OMEGA. when platinum black was not present.
Accordingly, it was confirmed that the coating of platinum black greatly
improved conductivity.
In this embodiment, ITO was used for electrode 1 and lead wire 2, NPI for
the insulating layer, and gold for the electrode surface coating material,
but it has already been stated that the material used shall not be limited
to these.
The process of producing the planar electrode of the invention is not
limited to the method described in this embodiment.
(Embodiment 2)
Next, the culture of nerve cells on the planar electrode is described.
On the planar electrode formed in Embodiment 1, cerebral visual cortex
cells of rats were cultured as the nerve cells.
Now, the culture method will be discussed in detail.
(a) Brains of fetuses of SD rats at 16-18 days of pregnancy were removed
and immersed in iced Hanks' Balanced Salt Solution (hereinafter called
"HBSS").
(b) From the brains in the iced HBSS, visual cortices were cut out and
transferred to Eagle's minimum essential medium (hereinafter called "MEM")
liquid.
(c) In the MEM liquid, the visual cortices were cut into as small pieces as
possible, 0.2 mm square at maximum.
(d) The visual cortices cut into small pieces were placed in centrifugal
tubes (test tubes for centrifugal separation), and after washing with HBSS
free from calcium and magnesium (hereinafter called "CMF-HBSS") three
times, they were dispersed in a suitable volume of the same liquid.
(e) In the centrifugal tubes of Step (d), a CMF-HBSS solution of trypsin
(0.25 wt %) was added to double the total volume. With gentle stirring,
enzymatic processes were allowed to take place while the solution was
incubated at 37.degree. C. for 15 to 20 minutes.
(f) DMEM/F-12 mixture medium in which Dulbecco modified Eagle's medium
(DMEM) and HamF-12 medium were mixed in a volume ratio of 1:1 and provided
with 10 vol. % of fetal cow serum (FCS) was added to the centrifugal tube
subjected to Step (e) to further double the total volume. With a Pasteur
pipette with a reduced diameter produced by fire-polishing the tip end
with a burner, gently repeating pipetting (about 20 times at maximum), the
cells were unravelled.
(g) Centrifugation was carried out for about 5 minutes at 9806.65
m/sec.sup.2 (that is, 1000 g). Upon completion of centrifugation, the
supernatant was discarded and the precipitate was suspended in DMEM/F-12
mixture medium containing FCS 5 vol. %.
(h) Step (g) was repeated two more times (a total of 3 times).
(i) The precipitate finally obtained was suspended in the DMEM/F-12 mixture
medium containing 5 vol. % FCS and using an erythrocytometer, the cell
concentration in the suspension liquid was measured. Using the similar
medium, the cell concentration was adjusted to be 2.times.10.sup.6 to
4.times.10.sup.6 cells/ml.
(j) In a well for cell culture formed by affixing a polystyrene cylinder
(inner diameter 22 mm, outer diameter 26 mm, height 8 mm) to the planar
electrode with the planar electrode center aligned with the polystyrene
cylinder center, 500 .mu.L of the DMEM/F-12 mixture medium containing 5
vol. % FCS was added in advance and heated in a CO.sub.2 incubator (air
content: 95 vol %; CO.sub.2 content: 5 vol %; relative humidity: 97%;
Temperature: 37.degree. C.).
(k) In the well of Step (j), 100 .mu.L of the suspension liquid with the
cell concentration adjusted was gently added and again let stand in the
CO.sub.2 incubator.
(l) Three days after the performance of Step (k), one half the medium was
replaced with a new one. For the replaced medium, the DMEM/F-12 mixture
medium not containing FCS was used.
(m) Thereafter, half of the medium was replaced in the similar manner every
4 to 5 days.
Over the series of these operations, nerve cells of cerebral visual
cortices of rats were cultured on the planar electrodes.
The cells grew successfully even on the insulating layer (NPI) and even on
the electrodes with platinum black deposited on them. Consequently, the
use of the electrodes suitably located for the stimulation electrodes or
recording electrodes enabled the simultaneous multi-point measurement of
the electrical activities of nerve cells.
FIGS. 4 and 5 show examples of electrical responses (potential changes) of
nerve cells at the electrodes located at suitable places recorded before
and after constant current stimulation of 100 .mu.A was provided over one
week at a frequency of 1 Hz via the electrodes suitably located in the
planar electrode of the invention. FIG. 4 shows the electrical responses
of the nerve cells before stimulation, while FIG. 5 shows the electrical
responses of the nerve cells after stimulation.
In addition, FIGS. 6 and 7 show examples of electrical responses of nerve
cells recorded before and after long-term stimulation was applied under
the same conditions as above, using a planar electrode whose surface is
not coated with Ni, Au, and platinum black. FIG. 6 shows the records of
electrical response of nerve cells before stimulation, while FIG. 7 shows
the records of electrical response of nerve cells after stimulation.
In FIGS. 4 through 7, the arrow marks show the artifacts generated as a
result of application of stimulation current and the arrow head shows a
potential change generated by electrical activities of nerve cells.
As is clear from FIG. 6, when the planar electrode whose surface is not
coated with Ni, Au, and platinum black is used, generation of artifacts is
great, while when the planar electrode of one embodiment of the invention
shown in FIG. 4 is used, generation of artifacts is suppressed.
As is clear from FIG. 7, when the planar electrode surface is not coated
with Ni, Au, and platinum black, generation of artifacts is greater after
than before stimulation; the electrical activities of the nerve cells are
hidden by the artifacts and measurement is disabled. In contrast, when the
planar electrode of one embodiment of the invention as shown in FIG. 5 is
used, same as in the case shown in FIG. 4, generation of artifacts is
suppressed and the electrical activities of the nerve cells are
successfully recorded.
There are many other methods than those described above for the culture of
nerve cells on the planar electrode of the invention.
The invention may be embodied in other specific forms without departing
from the spirit or essential characteristics thereof. The disclosed
embodiments are to be considered in all respects as illustrative and not
restrictive, the scope of the invention being indicated by the appended
claims rather than by the foregoing description and all changes which come
within the meaning and range of equivalency of the claims are intended to
be embraced therein.
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